PROJECT SUMMARY/ABSTRACT: Ribosomes are ribonucleoprotein particles that contain 50-80 different proteins and 3-4 RNAs. These complex machines catalyze protein synthesis in almost all cells in nature. The long-term goal of this project is to understand how eukaryotic ribosomes are assembled in vivo. We use the yeast Saccharomyces cerevisae, to facilitate molecular genetic, biochemical, and proteomic approaches. Production of ribosomes is tightly linked to cell growth and proliferation. Consequently dysregulation of ribosome biogenesis is linked to many diseases such as cancer or developmental disorders. Because pathways of ribosome biogenesis are very conserved, our studies in yeast will help understand mechanisms of regulation and dysregulation of ribosome production in humans. In addition to ribosomal proteins and rRNAs, more than 200 assembly factors are present in nascent eukaryotic ribosomes and are required for their assembly. Production of these complex ribonucleoprotein machines requires a dynamic series of remodeling steps in which protein-protein, protein-RNA, and RNA-RNA interactions are established and reconfigured to produce functional ribosomes. Because most assembly factors are predicted to play a structural rather than enzymatic role, we need to understand in more detail how the networks of interactions among them and with r proteins and pre-rRNAs are established and reorganized to drive assembly. Potential conformational switches of RNA-RNA interactions that are likely to play an important role to regulate the timing and fidelity of assembly also need to be explored in more detail. We focus on twelve assembly factors and nearby ribosomal proteins that exhibit physical and functional interactions with each other and with pre-rRNA in precursors to 60S ribosomal subunits. We want to understand how this interaction network enables formation of stable precursor particles able to undergo processing of 27S precursors to 5.8S and 25S ribosomal RNAs. We will use genetic selections and site-directed mutagenesis to generate mutations in these proteins or pre-rRNAs, designed to disrupt interactions, including potential conformational switches in pre-rRNAs. We will then employ a battery of assays, and adapt new ones, to investigate effects of these mutations on pre-rRNA folding and processing, and assembly of ribosomal proteins into preribosomes.